Amino acids produced by microorganisms find extensive use as feedstuff and food additives in the agriculture and food industry, as components of various nutrient mixtures for medical purposes and as reagents in the chemical and pharmaceutical industries.
Known in the art are methods for preparing strains which produce amino acids such as L-lysine, L-threonine, L-isoleucine, L-valine and the like by using various mutagens. The resulting mutant strains of microorganisms have specific genetically preconditioned defects in regulating metabolism and, due to such defects, they evolve into the nutrient medium, or produce specific amino acids. The required strains of microorganisms are produced by conventional methods based on the particular nutritive demand of a mutant (auxotrophy) or on resistance of a mutant to one or another structural analogue of an amino acid inhibiting the growth of the parental strain.
Amino acid production by known auxotrophic strains results from blocking the formation of a by-product or a coinhibitor amino acid, which participate in the negative control of the amino acid biosynthesis. Known are pantothenate auxotrophs of Escherichia coli, producing valine (Maas, W. K., Vogel, H. J., J. Bacteriol., v. 65, p. 388, 1953), homoserine-requiring strains of Corynebacterium glutamicumand Brevibacterium flavum, producing lysine (Nakayama et al., J. Gen. Appl. Microbiol., v. 7, p. 41, 1961), isoleucine-, threonine-, or homoserine-requiring mutants of Arthrobacter paraffineus and Corynebacterium hydrocarboclastus producing valine (U.S. Pat. No. 3,700,556).
Amino acid production by mutant strains resistant to structural analogues of amino acids results from destroying the negative regulation of the amino acid biosynthesis, i.e., feed-back inhibition of the key enzyme activity or repression of the corresponding enzyme's formation by the end products. Known are the S-2-aminoethyl-L-cysteine resistant mutant of genus Brevibacterium producing lysine (sano, K., Shiio, J. Gen. Appl. Microbiol., v. 16., p. 373, 1970, Shiio et al., J. Biochem., v. 68, p. 701, 1970), the amino-hydroxyvaleric acid (AHV) resistant mutant of Escherichia coli producing threonine (Shiio, I., Nakamoris, Agr. Biol. Chem., v. 33, p. 1152, 1969), AHV resistant mutant of the Brevibacterium producing threonine and the mutant of microorganisms belonging to the genus Brevibacterium or Corynebacterium, resistant to amino-hydroxyvaleric acid producing isoleucine (U.S. Pat. No. 3,767,529), mutant strains of the genera Brevibacterium and Corynebacterium having resistance to 2-thiasolalanine which produce valine (U.S. Pat. No. 3,893,888), mutant strains of Serratia marcescens resistant to isoleucine hydroxamate producing isoleucine (Kisumi etal, J. Bacteriol., v. 110, p. 761, 1972).
Amino acid-producing strains having a resistance to an amino acid analogue together with a nutrient requirement which increases their productivity are also known (U.S. Pat. No. 3,893,888). Such amino acid producers remain auxotrophic and can grow only on media containing specific additives.
Known in the art is also a method for preparing bacterial strains that produce amino acids which is based on the isolation of chromosome DNA fragments of a donor bacterium containing genes controlling the synthesis of a'selected amino acid, combining them with a multicopy plasmid DNA molecule by in vitro manipulation, and transforming a recipient strain with a hybrid DNA molecule to yield a bacterial strain possessing increased productivity (U.S. Pat. Nos. 4,278,765; 4,391,907). According to this method chromosomal DNA fragments are isolated from a strain a having a mutation which destroys the negative regulation of selected amino acid biosynthesis. The recipient strain may be a specially constructed strain or it may be the donor strain.
However no effective Escherichia coli strains producing isoleucine or valine have been obtained by this method. Therefore a need continues to exist for the development of a novel method for preparing amino acid producing strains.
Hitherto unknown are strains characterized by increasing production of an amino acid due to mutation in genes coding for the corresponding aminoacyl-tRNA synthetase. The aminoacyl-tRNA synthetases, or activating enzymes, are particularly crucial elements in the route leading from amino acids to proteins. These enzymes catalyze the formation of activated amino acids, that is their attachment to one or more specific tRNA. In most cases there is but one aminoacyl-tRNA synthetase for each amino acid. So, only conditionally expressed mutations may be obtained.
Mutants with altered aminoacyl-tRNA synthetases were described in several papers:
1. Roth, J. R., and Ames, B. N., J. Mol. Biol., v. 22, p. 325, 1966. PA0 2. Neidllardt, F. C., Bacteriol. Rev., v. 30, p. 701, 1966. PA0 3. Folk, W. R., and Berg, P., J. Bacteriol., v. 102, p. 193, 1970. PA0 4. Iaccarino, M., and Berg, P., J. Bacteriol., v. 105, p. 527, 1971. PA0 5. Johnson, E. M. et al, J. Bacteriol., v. 129, p. 66, 1979.
Some aminoacyl-tRNA synthetase mutations manifest themselves as auxotrophy. This is an unusual auxotrophy, because the defect is not in the formation of an amino acid, but in its utilization for protein synthesis. Also it was communicated, that phenotypic suppression of such auxotrophic mutations may arise as a result of additional mutations which increase the biosynthesis and intracellular concentration of the corresponding amino acid. However, amino acid producing strains constructed on the basis of auxotrophic aminoacyl-tRNA synthetase mutations and methods for their production were not known. Also, the role of such mutations in overproduction of amino acids by producer strains was not established.